In recent years, attention has been given to the development of recombinant poxvirus technology. Such poxvirus-based vectors are useful for a range of uses, for example generating immune responses such as vaccines, for the development of new vaccines, for delivery of desired proteins and for gene therapy. The advantages of these vectors include: (i) ease of generation and production; (ii) the large size of the genome permitting insertion of multiple genes, (iii) efficient delivery of genes to multiple cell types, including antigen-presenting cells; (iv) high levels of protein expression; (v) optimal presentation of antigens to the immune system; and (vi) the ability to elicit cell-mediated immune responses as well as antibody responses; and (vii) the long-term experience gained with using this vector in humans as a small pox vaccine.
Poxviruses can be genetically engineered to contain and express foreign DNA with or without impairing the ability of the virus to replicate. Such foreign DNA can encode a wide range of proteins, such as antigens that induce protection against one or more infectious agents, immune modulating proteins such as co-stimulatory molecules, or enzymatic proteins. For example, recombinant vaccinia viruses have been engineered to express immunizing antigens of herpesvirus, hepatitis B, rabies, influenza, human immunodeficiency virus (HIV), and other viruses (Kieny et al., Nature 312:163-6 (1984); Smith et al., Nature 302: 490-5 (1983); Smith et al., Proc. Natl. Acad. Sci. USA 80:7155-9 (1983); Zagury et al., Nature 326:249-50 (1987); Cooney et al., Lancet 337:567-72 (1991); Graham et al., J. Infect. Dis. 166:244-52 (1992), and have been shown to elicit immune responses against influenza virus, dengue virus, respiratory syncytial virus, and human immunodeficiency virus.
Poxviruses have also been used to generate immune reactions against tumor-associated antigens such as CEA, PSA and MUC.
Poxviruses are also attractive candidates for use in gene therapy for the delivery of genetic material into cells for therapeutic use. See U.S. Pat. No. 5,656,465. Compared to other systems such as retrovirus vectors (including lentiviral vectors), adenoviral vectors, and adeno-associated virus vectors, the large genome of poxviruses enables large genes to be inserted into pox-based vectors. Yet, because of the cytoplasmic nature of the virus integration of foreign DNA into a host cell's chromosomes will not occur.
Historically, poxviruses were used as a vaccine for protection against smallpox. Variola virus is the etiological agent of smallpox. During the smallpox era, overall mortality rates were approximately 30%. Vaccinia virus is a highly effective immunizing agent that enabled the global eradication of smallpox. The last naturally occurring case of smallpox occurred in Somalia in 1977. In May 1980, the World Health Assembly certified that the world was free of naturally occurring smallpox. As a result, smallpox vaccination was halted in the US, except for military personnel and laboratory workers. However, recent world events have resulted in a renewed interest in smallpox vaccination as a response to potential bioterrorism.
Vaccinia virus is the prototype of the genus Orthopoxvirus. It is a double-stranded DNA (deoxyribonucleic acid) virus that has a broad host range under experimental conditions but is rarely isolated from animals outside the laboratory (Fenner et al. Orthopoxviruses. San Diego, Calif.: Academic Press, Inc., 1989; Damaso et al., Virology 277:439-49 (2000)). Multiple strains of vaccinia virus exist that have different levels of virulence for humans and animals. For example, the Temple of Heaven and Copenhagen vaccinia strains are highly pathogenic among animals, whereas the NYCBOH strain, from which the Wyeth vaccine strain was derived, had relatively low pathogenicity (Fenner et al. Smallpox and its eradication. Geneva, Switzerland: World Health Organization, 1988). Dryvax,® the vaccinia (smallpox) vaccine currently licensed in the United States, is a lyophilized, live-virus preparation of infectious vaccinia virus (Wyeth Laboratories, Inc., Marietta, Pa.). However, Dryvax® is associated with adverse effects due to local or generalized vaccinia virus replication in children, the elderly and the immunosuppressed.
Attention has focused on attenuated orthopox such as NYVAC (U.S. Pat. No. 5,364,773) and modified vaccinia Ankara (MVA). MVA was derived from the Ankara vaccinia strain CVA,1 which was used in the 1950s as a smallpox vaccine. In 1958, attenuation experiments were initiated in the laboratory of Dr. Anton Mayr (University of Munich) comprising terminal dilution of CVA in chicken embryo fibroblast (CEF) cells that ultimately resulted in over 500 passages. The resulting MVA is an attenuated, replication-defective virus, which is restricted to replication primarily in avian cells.2 Comparison of the MVA genome to its parent, CVA, revealed 6 major deletions of genomic DNA (deletion I, II, III, IV, V, and VI), totaling 31,000 basepairs. (Meyer et al., J. Gen. Virol. 72:1031-8 (1991)). MVA has been administered to numerous animal species, including monkeys, mice, swine, sheep, cattle, horses and elephants with no local or systemic adverse effects.1,3,4 Over 120,000 humans have been safely vaccinated with MVA by intradermal, subcutaneous or intramuscular injections.3 MVA has also been reported to be avirulent among normal and immunosuppressed animals (Mayr et al., Zentralb. Bakteriol. 167:375-90 (1978). Accordingly, in addition to utility as a smallpox vaccine, these more attenuated strains are particularly attractive poxviruses for use as vectors for immune modulation and gene therapy.
One of the main advantages of poxviruses as vectors is the large size of their genomes, which permits the insertion of a wide range of genetic material including multiple genes (i.e., as a multivalent vector). However, the genetic material must be inserted at an appropriate site within the pox genome for the recombinant virus to remain viable. Thus, the genetic material must be inserted at a site in the viral DNA which is non-essential.
Accordingly, it is desirable to identify specific sites within a poxvirus genome that can accommodate insertion of foreign DNA, while retaining the ability to infect foreign cells and express that DNA, while maintaining the desired immunogenicity and diminished virulence. Certain insertion sites are known in different poxviruses. For example, as described above, MVA contains 6 natural deletion sites, which have been demonstrated to serve as insertion sites. See e.g. U.S. Pat. No. 5,185,146, and U.S. Pat. No. 6,440,422. However, these insertion sites are only found in one specific strain of vaccinia, MVA. It would be desirable to identify insertion sites which are more broadly represented, i.e. are present in other attenuated strains, other vaccinia strains such as CVA, Wyeth and other vaccinia strains in addition to MVA, as well as present in other poxvirus genomes.
Accordingly, there remains a need in the art for improved pox vectors containing novel insertion sites, for use for example for parenteral immunization, as a vector system, or in the active or inactivated form as an adjuvant or as a regulator of the unspecific components of the immune system.